PROJECT SUMMARY Glioblastoma is a lethal primary brain tumor with limited treatment options. The current standard therapy with combined chemoradiation therapy (chemoRT) with temozolomide (TMZ), an alkylating agent offers a median survival of only 16-18 months. There is an urgent need for novel therapies especially those that can overcome resistance to chemoRT. Cancer cells develop complex resistance mechanisms that enable them to survive the effects of conventional therapies. One such evolutionally conserved mechanism is the heat shock response (HSR) which can deploy several diverse defense processes in the setting of adverse environmental conditions. The HSR is mediated by heat shock proteins (HSP), a class of molecular chaperones that shuttle and configure client oncoproteins into proper functional states. In preliminary studies, we show that onalespib, a novel long acting inhibitor of heat shock protein 90 (Hsp90), a critical mediator of the HSR in cancer cells, blocked tumor growth, invasion and angiogenesis in gliomas suggesting the potential for a strong independent antitumor effect. Relevant to this proposal, onalespib sensitizes glioma cells to TMZ and RT in patient-derived (PDX) cell lines and in a zebrafish and mouse intracranial glioma animal models. Based on these data, in this grant submission, we propose a phase I clinical trial through the NCI-funded Adult Brain Tumor Consortium to identify the maximum tolerated dose of the combination of onalespib with chemoRT in adults with newly diagnosed GBM. We also propose to conduct key correlative tissue and plasma studies through the following specific aims: Aim 1 will identify the MTD of onalespib in combination of chemoRT and adjuvant temozolomide and will determine whether onalespib can cross the blood brain barrier and achieve sufficient concentrations in enhancing and non-enhancing glioma tissue compared with plasma levels. Aim 2 will determine the pharmacodynamic effects of onalespib by assessing whether onalespib can inhibit Hsp90, its target, in human GBM tissue obtained in this trial and whether this inhibition can affect its chaperone oncoprotein clients particularly those relevant to DNA repair and cell survival against the effects of RT and TMZ. In Aim 3, we will conduct co-clinical trials using PDX intracranial glioma models and organotypic human glioma slices to determine the mechanisms of sensitivity and resistance to onalespib effects to provide insights that can help modify the subsequent phase II trial. This is the first human trial of an Hsp90 inhibitor in brain tumors and the first to combine an Hsp90 inhibitor with chemo- and radiation therapy against GBM. Successful completion of this trial will enable us to proceed to a Phase II efficacy trial (approved by NRG oncology) and will provide comprehensive PK and PD data that can help the development of onalespib and Hsp90 inhibitors in other malignancies.